For scientific, regulatory, or other applications, many persons, institutions, and agencies often require analyses of samples to determine whether such small molecule and ionic analytes are present. Examples of such analyses include the determination of metal ions in sea water to understand the processes of chemical oceanography; determination of toxic materials such as Hg(II), Ni(II), CN.sup.-, or HS.sup.- in groundwater or wastewater; detection of the corrosion of metal alloys by the presence of Co(II), Zn(II), or Cu(II) in condensates; or the presence of metals in lubricating oil as an indicator of machinery wear and incipient failure.
Many methods are known in the art for such analyses. For metal ions, such methods include graphite furnace atomic absorption spectrophotometry, inductively coupled plasma atomic emission spectroscopy and mass spectroscopy, various electrochemical means, and fluorescence spectroscopy using metallofluorescent indicators. For common anionic analytes there are fewer techniques available; they include ion chromatography, mass spectrometry, and electrochemical means.
Most of these techniques involve analysis of single or multiple discrete samples in a specialized instrument which may not be close to the sample. This is a particular drawback for analytical tasks that require a continuous or quasicontinuous determination of the analyte with real time readout of the result; require samples to be collected from remote, inaccessible, or hazardous environments; or require such extensive sampling that it is prohibitively costly. For many of these methods a sensor capable of remotely, continuously, and selectively monitoring the analyte of interest in situ is required, and subsequent reporting of the results of the analysis back to the operator in real time.
Improvements have been made in the development of fluorescence-based sensors for a variety of applications (Thompson, R. B. (1991) in Topics in Fluorescence Spectroscopy, Vol. 2: Principles, Lakowicz, J. R. (Ed.) Plenum Press, NY; Wolfbeis, O. S. (Ed.) (1992) Fiber Optic Chemical Sensors and Biosensors Vols. I and II, CRC Press, Boca Raton, Fla.; Lakowicz, J. R., and Thompson, R. B. (Eds.) (1993) Proc. of the SPIE Conference on Advances in Fluorescence Sensing Technology Vol. 1885, Society of Photooptical Instrumentation Engineers, Bellingham, Wash.) A central issue in the development of such sensors has been the means of transduction, whereby the presence or relative amount of the chemical analyte is transduced as a change in the fluorescence which may be quantitated. Thus, workers in the field have mainly transduced analyte levels as changes in fluorescence intensity (Thompson (1991); Saari, L. A., and Seitz, W. R. (1982) Anal. Chem. 54, 821; Thompson, R. B. and Ligler, F. S. (1991) in Biosensors with Fiber Optics., Wise, D., and Wingard, L. (Eds.) pp. 111-138, Humana Press, Clifton, N.J.), or ratios of fluorescence intensity at two different wavelengths (Tsien, R. Y. (1989) Ann. Rev. Neurosci. 12, 227; Opitz, N., and Lubbers, D. W. (1984) Adv. Exp. Med. Biol. 180, 757; Thompson, R. B. and Jones, E. R. (1993) Anal. Chem. 65, 730-4; and U.S. Pat. No. 5,545,517).
The ratio approach has proven particularly popular because it is robust in avoiding many of the artifacts associated with simple intensity measurements. The major limitation of the ratio approach has been the limited number of ratiometric fluorescent indicators (Haugland, R. P. (1992) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, Oreg.).
Recently, many groups have shown that transducing the level of analyte as a change in fluorescence lifetime is a robust and flexible approach to optical sensing (Demas, J. N., (1994) in Topics in Fluorescence Spectroscopy, Vol. 4: Probe Design and Chemical Sensing, Lakowicz, J. R. (Ed.) Plenum Press, NY; Lippitsch, M. E., Pusterhofer, J., Leiner, M. J. P. and Wolfbeis, O. S. (1988) Anal. Chim. Acta 205, 1-6; Keating, S. M., and Wensel, T. G. (1991) Biophys. J. 59, 186-202; Lakowicz, J. R., Szmacinski, H., and Karakelle, M. (1993) Anal. Chim. Acta 272, 179-186; Szmacinski, H., and Lakowicz, J. R. (1993) Anal. Chem. 65, 1668-74; Lakowicz, J. R. (1992) Laser Focus World 28(5) 60-80; Thompson, R. B. and Patchan, M. W. (1993) in Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V. Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.; Ozinskas, A. J., Malak, H., Joshi, J., Szmacinski, H., Britz, J., Thompson, R. B., Koen, P. A., and Lakowicz, J. R. (1993) Anal. Biochem. 213, 264-270). Lifetime-based sensing can exhibit a dynamic range of greater than five orders of magnitude in analyte concentration (Szmacinski, H., and Lakowicz, J. R. (1993) Anal. Chem. 65, 1668-74; Thompson, R. B. and Patchan, M. W. (1993) in Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V. Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.). Lifetime-based sensing has been adapted to and has particular advantages for fiber optic sensors.
The development of fluorescence-based fiber optic biosensors is well documented. In particular, optical designs which optimize fluorescence measurements through optical fiber are described (U.S. Pat. No. 5,141,132), a sensor suited for the determination of anesthetics and other lipid-soluble analytes (U.S. Pat. No. 5,094,819), and an optical design which optimizes the sensitivity of so-called evanescent-wave sensors (U.S. Pat. No. 5,061,897) have been described. The advantages of fiber optic sensors for remote, continuous monitoring of analytes in environments that are hazardous or inaccessible are well known (Thompson, R. B. (1991) in Topics in Fluorescence Spectroscopy, Vol. 2: Principles, Lakowicz, J. R. (Ed.) Plenum Press, NY; Wolfbeis, O. S. (Ed.) (1992) Fiber Optic Chemical Sensors and Biosensors Vols. I and II, CRC Press, Boca Raton, Fla.; Thompson, R. B. and Ligler, F. S. (1991) in Biosensors with Fiber Optics. Wise, D., and Wingard, L. (Eds.) pp. 111-138, Humana Press, Clifton, N.J.).
Recently, a fiber optic biosensor for metal ions in aqueous solution that takes advantage of the very selective binding of particular metals by the enzyme carbonic anhydrase II from mammalian erythrocytes has been described. (Thompson, R. B. and Jones, E. R. (1993) Anal. Chem. 65, 730-4; Thompson, R. B. and Patchan, M. N. (1993) in Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V. Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.; and U.S. Pat. No. 5,545,517). It was shown that metal-dependent binding of a fluorescent aryl sulfonamide inhibitor, dansylamide, could be transduced as a change in the ratio of fluorescence emission intensities at two different wavelengths (450 and 550 nanometers (R. B. Thompson and E. R. Jones, Anal. Chem. 65: 730-4 (1993)), or changes in the fractional contributions of two fluorescence lifetimes arising from the bound and free forms of the dansylamide (see FIG. 1) (R. B. Thompson and M. W. Patchan, J. Fluorescence 5:123-30 (1995)). These fluorescence observables are related to the fraction of enzyme with dansylamide bound to it, which is equal to the fraction of enzyme with Zn(II) in its active site and simply related to the concentration of the analyte Zn(II) by the law of mass action. These methods demonstrate rapid determination of Zn(II) at nanomolar levels in aqueous solutions.
However, dansylamide has some properties which are suboptimal. It must be excited by ultraviolet light at approximately 330 nanometers, a regime at which typical optical fiber exhibits high attenuation and background fluorescence, and for which available light sources are relatively inconvenient and expensive. While it is desirable to find a fluorescent inhibitor akin to dansylamide but excitable in the visible or near infrared, searches of the extensive literature of arylsulfonamide inhibitors of mammalian carbonic anhydrases failed to elicit a compound with the desired properties (Bar, D. (1963) Act. Pharm. 15, 1-44). Colored inhibitors such as azosulfamide are known in the art (Krebs Biochem. J., 43:525-528 (1948)). However, their use as an additional ligand on an active site metal ion or ligand to quench by energy transfer is first demonstrated by the present invention. While this inhibitor, azosulfamide, does not fluoresce significantly itself, the present invention demonstrates transduction of zinc-dependent binding to the enzyme as a change in fluorescence lifetime using the technique of energy transfer.
Ullman (U.S. Pat. No 4,261,968) teaches a fluorescence immunoassay wherein a fluorescent-labeled antibody is brought in contact with a sample containing antigen (the analyte) and a quencher-labeled antigen; the unlabeled antigen (of unknown concentration) and labeled antigen (of known concentration) compete for the antibody recognition site. The fluorescence emission of the label is quenched to a degree inversely proportional to the unlabeled antigen concentration, and thus serves as a measure thereof. Ullman does not teach analysis of metal ions or small anions, or the use of a ligand whose binding is metal-ion-dependent, or the use of lifetime-based sensing as in the present invention.
Fluorescence resonance energy transfer is a dipole-dipole interaction described by Forster (Forster, Th. (1948) Ann. Physik 2, 55-75). Forster's theory is very well-established, with thousands of examples in the literature of its predictive power. The rate of energy transfer K.sub.T is a function of the distance between donor and acceptor r, the refractive index of the medium n, the degree of energy overlap J between the emission spectrum of the donor and the absorbance spectrum of the acceptor, the emissive rate of the donor in the absence of acceptor .lambda..sub.d, and the relative orientation between the donor and acceptor dipoles .kappa..sup.2 : EQU K.sub.T =(r.sup.-6 J.kappa..sup.2 n.sup.-4.lambda..sub.d).times.8.71.times.10.sup.23 sec.sup.-1
The rate of energy transfer can be simply expressed in terms of a Forster distance R.sub.0, which is the distance at which the rates of emission and energy transfer are equal, and the lifetime of the donor .tau..sub.D : ##EQU1##
A labeled macromolecule such as carbonic anhydrase, wherein the donor fluorophore is approximately at its Forster distance from the azosulfamide in the active site, would exhibit a more rapid apparent decay of its fluorescence due to energy transfer; energy transfer is thus a quenching mechanism, and a decrease in fluorescence intensity should be observed as well. It is well known that by placing the donor much closer to the active site, essentially quantitative quenching results, whereas if the donor is much further away, the quenching is modest due to the sixth power dependence.
It has been demonstrated in the present invention that positioning of the fluorescent donor moiety on the macromolecule at particular distances from a metal ion bound to the macromolecule or an inhibitor bound to the metal ion optimizes the response of the assay.
Thus, it has been found in the present invention, that a sensor, which we term `fluorescence-based biosensor", transduces the presence or level of the metal ion or ligand as a change in the fluorescence of an indicator phase, which can be measured through a length of optical fiber with the indicator phase at the distal end in contact with a sample containing the metal ion or ligand at the proximal end.
This invention demonstrates for the first time the determination of metal ions and ligands such as anions using a photoluminescent energy transfer mechanism employing a macromolecule. This invention also demonstrates for the first time the importance of controlling the proximity of the photoluminescent-donor-label to the metal ion or ligand in making the determinations.